Positional sequencing by hybridization

ABSTRACT

This invention is directed to methods and reagents useful for sequencing nucleic acid targets utilizing sequencing by hybridization technology comprising probes, arrays of probes and methods whereby sequence information is obtained rapidly and efficiently in discrete packages. That information can be used for the detection, identification, purification and complete or partial sequencing of a particular target nucleic acid. When coupled with a ligation step, these methods can be performed under a single set of hybridization conditions. The invention also relates to the replication of probe arrays and methods for making and replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5′- and/or 3′-overhangs.

REFERENCE TO RELATED APPLICATIONS

[0001] This is a Continuation of copending application Ser. No. 08/470,832 filed Jun. 06, 1995, which is a divisional of application Ser. No. 08/110,691 filed Aug. 23, 1993, which is a Continuation-In-Part of application Ser. No. 07/972,012 filed Nov. 06, 1992.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to methods for sequencing nucleic acids by positional hybridization and to procedures combining these methods with more conventional sequencing techniques and with other molecular biology techniques including techniques utilized in PCR (polymerase chain reaction) technology. Useful applications include the creation of probes and arrays of probes for detecting, identifying, purifying and sequencing target nucleic acids in biological samples. The invention is also directed to novel methods for the replication of probe arrays, to the replicated arrays, to diagnostic aids comprising nucleic acid probes and arrays useful for screening biological samples for target nucleic acids and nucleic acid variations.

[0004] 2. Description of the Background

[0005] Since the recognition of nucleic acid as the carrier of the genetic code, a great deal of interest has centered around determining the sequence of that code in the many forms in which it is found. Two landmark studies made the process of nucleic acid sequencing, at least with DNA, a common and relatively rapid procedure practiced in most laboratories. The first describes a process whereby terminally labeled DNA molecules are chemically cleaved at single base repetitions (A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA 74:560-564, 1977). Each base position in the nucleic acid sequence is then determined from the molecular weights of fragments produced by partial cleavages. Individual reactions were devised to cleave preferentially at guanine, at adenine, at cytosine and thymine, and at cytosine alone. When the products of these four reactions are resolved by molecular weight, using, for example, polyacrylamide gel electrophoresis, DNA sequences can be read from the pattern of fragments on the resolved gel.

[0006] The second study describes a procedure whereby DNA is sequenced using a variation of the plus-minus method (F. Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-67, 1977). This procedure takes advantage of the chain terminating ability of dideoxynucleoside triphosphates (ddNTPs) and the ability of DNA polymerase to incorporate ddNTP with nearly equal fidelity as the natural substrate of DNA polymerase, deoxynucleosides triphosphates (dNTPs). A primer, usually an oligonucleotide, and a template DNA are incubated together in the presence of a useful concentration of all four dNTPs plus a limited amount of a single ddNTP. The DNA polymerase occasionally incorporates a dideoxynucleotide which terminates chain extension. Because the dideoxynucleotide has no 3′-hydroxyl, the initiation point for the polymerase enzyme is lost. Polymerization produces a mixture of fragments of varied sizes, all having identical 3′ termini. Fractionation of the mixture by, for example, polyacrylamide gel electrophoresis, produces a pattern which indicates the presence and position of each base in the nucleic acid. Reactions with each of the four ddNTPs allows one of ordinary skill to read an entire nucleic acid sequence from a resolved gel.

[0007] Despite their advantages, these procedures are cumbersome and impractical when one wishes to obtain megabases of sequence information. Further, these procedures are, for all practical purposes, limited to sequencing DNA. Although variations have developed, it is still not possible using either process to obtain sequence information directly from any other form of nucleic acid.

[0008] A new method of sequencing has been developed which overcomes some of the problems associated with current methodologies wherein sequence information is obtained in multiple discrete packages. Instead of having a particular nucleic acid sequenced one base at a time, groups of contiguous bases are determined simultaneously by hybridization. There are many advantages including increased speed, reduced expense and greater accuracy.

[0009] Two general approaches of sequencing by hybridization have been suggested. Their practicality has been demonstrated in pilot studies. In one format, a complete set of 4^(n) nucleotides of length n is immobilized as an ordered array on a solid support and an unknown DNA sequence is hybridized to this array (K. R. Khrapko et al., J. DNA Sequencing and Mapping 1:375-88, 1991). The resulting hybridization pattern provides all n-tuple words in the sequence. This is sufficient to determine short sequences except for simple tandem repeats.

[0010] In the second format, an array of immobilized samples is hybridized with one short oligonucleotide at a time (Z. Strezoska et al., Proc. Natl. Acad. Sci. USA 88:10,089-93, 1991). When repeated 4^(n) times for each oligonucleotide of length n, much of the sequence of all the immobilized samples would be determined. In both approaches, the intrinsic power of the method is that many sequenced regions are determined in parallel. In actual practice the array size is about 10⁴ to 10⁵.

[0011] Another powerful aspect of the method is that information obtained is quite redundant, especially as the size of the nucleic acid probe grows. Mathematical simulations have shown that the method is quite resistant to experimental errors and that far fewer than all probes are necessary to determine reliable sequence data (P. A. Pevzner et al., J. Biomol. Struc. & Dyn. 9:399-410, 1991; W. Bains, Genomics 11:295-301, 1991).

[0012] In spite of an overall optimistic outlook, there are still a number of potentially severe drawbacks to actual implementation of sequencing by hybridization. First and foremost among these is that 4^(n) rapidly becomes quite a large number if chemical synthesis of all of the oligonucleotide probes is actually contemplated. Various schemes of automating this synthesis and compressing the products into a small scale array, a sequencing chip, have been proposed.

[0013] A second drawback is the poor level of discrimination between a correctly hybridized, perfectly matched duplexes, and an end mismatch. In part, these drawbacks have been addressed at least to a small degree by the method of continuous stacking hybridization as reported by a Khrapko et al. (FEBS Lett. 256:118-22, 1989). Continuous stacking hybridization is based upon the observation that when a single-stranded oligonucleotide is hybridized adjacent to a double-stranded oligonucleotide, the two duplexes are mutually stabilized as if they are positioned side-to-side due to a stacking contact between them. The stability of the interaction decreases significantly as stacking is disrupted by nucleotide displacement, gap, or terminal mismatch. Internal mismatches are presumably ignorable because their thermodynamic stability is so much less than perfect matches. Although promising, a related problem arises which is the inability to distinguish between weak but correct duplex formation, and simple background such as non-specific adsorption of probes to the underlying support matrix.

[0014] A third drawback is that detection is monochromatic. Separate sequential positive and negative controls must be run to discriminate between a correct hybridization match, a mis-match, and background.

[0015] A fourth drawback is that ambiguities develop in reading sequences longer than a few hundred base pairs on account of sequence recurrences. For example, if a sequence the same length of the probe recurs three times in the target, the sequence position cannot be uniquely determined. The locations of these sequence ambiguities are called branch points.

[0016] A fifth drawback is the effect of secondary structures in the target nucleic acid. This could lead to blocks of sequences that are unreadable if the secondary structure is more stable than occurs on the complimentary strand.

[0017] A final drawback is the possibility that certain probes will have anomalous behavior and for one reason or another, be recalcitrant to hybridization under whatever standard sets of conditions ultimately used. A simple example of this is the difficulty in finding matching conditions for probes rich in G/C content. A more complex example could be sequences with a high propensity to form triple helices. The only way to rigorously explore these possibilities is to carry out extensive hybridization studies with all possible oligonucleotides of length n, under the particular format and conditions chosen. This is clearly impractical if many sets of conditions are involved.

[0018] Among the early publication which appeared discussing sequencing by hybridization, E. M. Southern (PCT application no. WO 89/10977, published Nov. 16, 1989; which is hereby specifically incorporated by reference), described methods whereby unknown, or target, nucleic acids are labeled, hybridized to a set of nucleotides of chosen length on a solid support, and the nucleotide sequence of the target determined, at least partially, from knowledge of the sequence of the bound fragments and the pattern of hybridization observed. Although promising, as a practical matter, this method has numerous drawbacks. Probes are entirely single-stranded and binding stability is dependant upon the size of the duplex. However, every additional nucleotide of the probe necessarily increases the size of the array by four fold creating a dichotomy which severly restricts its plausible use. Further, there is an inability to deal with branch point ambiguities or secondary structure of the target, and hybridization conditions will have to be taylored or in some way accounted for for each binding event.

[0019] R. Drmanac et al. (U.S. Pat. No. 5,202,231; which is specifically incorporated by reference) is directed to methods for sequencing by hybridization using sets of oligonucleotide probes with randon sequences. These probes, although useful, suffer from some of the same drawbacks as the methodology of Southern (1989), and like Southern, fail to recognize the advantages of stacking interactions.

[0020] K. R. Khrapko et al. (FEBS Lett. 256:118-22, 1989; and J. DNA Sequencing and Mapping 1:357-88, 1991) attempt to address some of these problems using a technique referred to as continuous stacking hybridization. With continuous stacking, conceptually, the entire sequence of a target nucleic acid can be determined. Basically, the target is hybridized to an array of probes, again single-stranded, denatured from the array, and the dissociation kinetics of denaturation analyzed to determine the target sequence. Although also promising, discrimination between matches and mis-matches (and simple background) is low, and further, as hybridization conditions are inconstant for each duplex, discrimination becomes increasingly reduced with increasing target complexity.

SUMMARY OF THE INVENTION

[0021] The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new methods for rapidly and accurately determining the nucleotide sequence of a nucleic acid by the herein described methods of positional sequencing by hybridization.

[0022] One embodiment of the invention is directed to arrays of R⁴ different nucleic acid probes wherein each probe comprises a double-stranded portion of length D, a terminal single-stranded portion of length S, and a random nucleotide sequence within the single-stranded portion of length R. These arrays may be bound to solid supports and are useful for determining the nucleotide sequence of unknown nucleic acids and for the detection, identification and purification of target nucleic acids in biological samples.

[0023] Another embodiment of the invention is directed to methods for creating arrays of probes comprising the steps of synthesizing a first set of nucleic acids each comprising a constant sequence of length C at the 3′-terminus, and a random sequence of length R at the 5′-terminus, synthesizing a second set of nucleic acids each comprising a sequence complimentary to the constant sequence of the first nucleic acid, and hybridizing the first set with the second set to form the array.

[0024] Another embodiment of the invention is directed to methods for creating arrays of probes comprising the steps of synthesizing a set of nucleic acids each containing a random internal sequence of length R flanked by the cleavage sites of a restriction enzyme, synthesizing a set of primers each compliementary to a non-random sequence of the nucleic acid, hybridizing the two sets together to form hybrids, extending the sequence of the primer by polymerization using the nucleic acid as a template, and cleaving the hybrids with the restriction enzyme to form an array of probes with a double-stranded portion and a single-stranded portion and with the random sequence within the single stranded portion.

[0025] Another embodiment of the invention is directed to replicated arrays and methods for replicating arrays of probes, preferably on a solid support, comprising the steps of synthesizing an array of nucleic acids each comprising a constant sequence of length C at a 3′-terminus and a random sequence of length R at a 5′-terminus, fixing the array to a first solid support, synthesizing a set of nucleic acids each comprising a sequence complimentary to the constant region of the array, hybridizing the nucleic acids of the set with the array, enzymatically extending the nucleic acids of the set using the random sequences of the array as templates, denaturing the set of extended nucleic acids, and fixing the denatured nucleic acids of the set to a second solid support to create the replicated array of probes. The replicated array may be single-stranded or double-stranded, it may be fixed to a solid support or free in solution, and it is useful for sequencing, detecting or simply identifying target nucleic acids.

[0026] The array is also useful for the purification of nucleic acid from a complex mixture for later identification and/or sequencing. A purification array comprises sufficient numbers of probes to hybridize and thereby effectively capture the target sequences from a complex sample. The hybridized array is washed to remove non-target nucleic acids and any other materials which may be present and the target sequences eluted by denaturing. From the elution, purified or semi-purified target sequences are obtained and collected. This collection of target sequences can then be subjected to normal sequencing methods or sequenced by the methods described herein.

[0027] Another embodiment of the invention is directed to nucleic acid probes and methods for creating nucleic acid probes comprising the steps of synthesizing a plurality of single-stranded first nucleic acids and a plurality of longer single-stranded second nucleic acids wherein each each second nucleic acid comprises a random terminal sequence and a sequence complimentary to a sequence of the first nucleic acids, hybridizing the first nucleic acids to the second to form partial duplexes having a double-stranded portion and a single-stranded portion with the random sequence within the single-stranded portion, hybridizing a target nucleic acid to the partial duplexes, optionally ligating the hybridized target to the first nucleic acid of the partial duplexes, isolating the second nucleic acid from the ligated duplexes, synthesizing a plurality of third nucleic acids each complimentary to the constant sequence of the second nucleic acid, and hybridizing the third nucleic acids with the isolated second nucleic acids to create the nucleic acid probe. Alternatively, after formation of the partial duplexes, the target is ligated as before and hybridized with a set of oligonucleotides comprising random sequences. These oligonucleotides are ligated to the second nucleic acid, the second nucleic acid is isolated, another plurality of first nucleic acids are synthesized, and the first nucleic acids are hybridized to the oligonucleotide ligated second nucleic acids to form the probe. Ligation allows for hybridization to be performed under a single set of hybridization conditions. Probes may be fixed to a solid support and may also contain enzyme recognition sites within their sequences.

[0028] Another embodiment of the invention is directed to diagnostic aids and methods utilizing probe arrays for the detection and identification of target nucleic acids in biological samples and to methods for using the diagnostic aids to screen biological samples. Diagnostic aids as described are also useful for the purification of identified targets and, if desired, for their sequencing. These aids comprise probes, solid supports, labels, necessary reagents and the biological samples.

[0029] Other advantages of the invention are set forth in part in the description which follows, and in part, will be obvious from this description, or may be learned from the practice of this invention. The accompanying drawings which are incorporated in and constitute a part of this specification, illustrate and, together with this description, serve to explain the principle of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 Energetics of stacking hybridization. Structures consist of a long target and a probe of length n. The top three sample are ordinary hybridization and the bottom three are stacking hybridization.

[0031]FIG. 2 (A) The first step of the basic scheme for positional sequencing by hybridization depicting the hybridization of target nucleic acid with probe forming a 5′ overhang of the target. (B) The first step of the alternate scheme for positional sequencing by hybridization depicting the hybridization of target nucleic acid with probe forming a 3′ overhang of the probe.

[0032]FIG. 3 Graphic representation of the ligation step of positional sequencing by hybridization wherein hybridization of the target-nucleic acid produces (A) a 5′ overhang or (B) a 3′ overhang.

[0033]FIG. 4 Preparation of a random probe array.

[0034]FIG. 5 Single nucleotide extension of a probe hybridized with a target nucleic acid using DNA polymerase and a single dideoxynucleotide.

[0035]FIG. 6 Preparation of a nested set of targets using labeled target nucleic acids partially digested with exonuclease III.

[0036]FIG. 7 Determination of positional information using the ratio of internal label to terminal label.

[0037]FIG. 8 (A) Extension of one strand of the probe using a hybridized target as template with a single deoxynucleotide. (B) Hybridization of target with a fixed probe followed by ligation of probe to target.

[0038]FIG. 9 Four color analysis of sequence extensions of the 3′ end of a probe using three labeled nucleoside triphosphates and one unlabeled chain terminator.

[0039]FIG. 10 Extension of a nucleic acid probe by ligation of a pentanucleotide 3′ blocked to prevent polymerization.

[0040]FIG. 11 Preparation of a customized probe containing a 10 base pair sequence that was present in the original target nucleic acid.

[0041]FIG. 12 Graphic representation of the general procedure of positional sequencing by hybridization.

[0042]FIG. 13 Graphical representation of the ligation efficiency of positional sequencing. Depicted is the relationship between the amount of label remaining over the total amounts of label in the reaction, verses NaCl concentration.

[0043]FIG. 14 A diagrammatic representation of the construction of a complimentary array of master beads.

DESCRIPTION OF THE INVENTION

[0044] The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new methods and probes, new diagnostic aids and methods for using the diagnostic aids, and new arrays and methods for creating arrays of probes to detect, identify, purify and sequence target nucleic acids. Nucleic acids of the invention include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be isolated from natural sources, recombinantly produced, or artificially synthesized. Preferred embodiments of the present invention is probe synthesized using traditional chemical synthesis, using the more rapid polymerase chain reaction (PCR) technology, or using a combination of these two methods.

[0045] Nucleic acids of the invention further encompass polyamide nucleic acid (PNA) or any sequence of what are commonly referred to as bases joined by a chemical backbone that have the ability to base pair or hybridize with a complimentary chemical structure. The bases of DNA, RNA, and PNA are purines and pyrimidines linearly linked to a chemical backbone. Common chemical backbone structures are deoxyribose phosphate and ribose phosphate. Recent studies demonstrated that a number of additional structures may also be effective, such as the polyamide backbone of PNA (P. E. Nielsen et al., Sci. 254:1497-1500, 1991).

[0046] The purines found in both DNA and RNA are adenine and guanine, but others known to exist are xanthine, hypoxanthine, 2- and 1-diaminopurine, and other more modified bases. The pyrimidines are cytosine, which is common to both DNA and RNA, uracil found predominantly in RNA, and thymidine which occurs exclusively in DNA. Some of the more atypical pyrimidines include methylcytosine, hydroxymethyl-cytosine, methyluracil, hydroxymethyluracil, dihydroxypentyluracil, and other base modifications. These bases interact in a complimentary fashion to form base-pairs, such as, for example, guanine with cytosine and adenine with thymidine. However, this invention also encompasses situations in which there is nontraditional base pairing such as Hoogsteen base pairing which has been identified in certain tRNA molecules and postulated to exist in a triple helix.

[0047] One embodiment of the invention is directed to a method for determining a nucleotide sequence by positional hybridization comprising the steps of (a) creating a set of nucleic acid probes wherein each probe has a double-stranded portion, a single-stranded portion, and a random sequence within the single-stranded portion which is determinable, (b) hybridizing a nucleic acid target which is at least partly single-stranded to the set of nucleic acid probes, and (c) determining the nucleotide sequence of the target which hybridized to the single-stranded portion of any probe. The set of nucleic acid probes and the target nucleic acid may comprise DNA, RNA, PNA, or any combination thereof, and may be derived from natural sources, recombinant sources, or be synthetically produced. Each probe of the set of nucleic acid probes has a double-stranded portion which is preferably about 10 to 30 nucleotides in length, a single-stranded portion which is preferably about 4 to 20 nucleotides in length, and a random sequence within the single-stranded portion which is preferably about 4 to 20 nucleotides in length and more preferably about 5 nucleotides in length. A principle advantage of this probe is in its structure. Hybridization of the target nucleic acid is encouraged due to the favorable thermodynamic conditions established by the presence of the adjacent double-strandedness of the probe. An entire set of probes contains at least one example of every possible random nucleotide sequence.

[0048] By way of example only, if the random portion consisted of a four nucleotide sequence (R=4) of adenine, guanine, thymine, and cystosine, the total number of possible combinations (4^(R)) would be 4⁴ or 256 different nucleic acid probes. If the number of nucleotides in the random sequence was five, the number of different probes within the set would be 4⁵ or 1,024. This becomes a very large number indeed when considering sequences of 20 nucleotides or more.

[0049] However, to determine the complete sequence of a nucleic acid target, the set of probes need not contain every possible combination of nucleotides of the random sequence to be encompassed by the method of this invention. This variation of the invention is based on the theory of degenerated probes proposed by S. C. Macevicz (International Patent Application, US89-04741, published 1989, and herein specifically incorporated by reference). The probes are divided into four subsets. In each, one of the four bases is used at a defined number of positions and all other bases except that one on the remaining positions. Probes from the first subset contain two elements, A and non-A (A=adenosine). For a nucleic acid sequence of length k, there are 4(2^(k)-1), instead of 4^(k) probes. Where k=8, a set of probes would consist of only 1020 different members instead of the entire set of 65,536. The savings in time and expense would be considerable. In addition, it is also a method of the present invention to utilize probes wherein the random nucleotide sequence contains gapped segments, or positions along the random sequence which will base pair with any nucleotide or at least not interfere with adjacent base pairing.

[0050] Hybridization between complimentary bases of DNA, RNA, PNA, or combinations of DNA, RNA and PNA, occurs under a wide variety of conditions such as variations in temperature, salt concentration, electrostatic strength, and buffer composition. Examples of these conditions and methods for applying them are described in Nucleic Acid Hybridization: A Practical Approach (B. D. Hames and S. J. Higgins, editors, IRL Press, 1985), which is herein specifically incorporated by reference. It is preferred that hybridization takes place between about 0° C. and about 70° C., for periods of from about 5 minutes to hours, depending on the nature of the sequence to be hybridized and its length. For example, typical hybridization conditions for a mixture of two 20-mers is to bring the mixture to 68° C. and let cool to room temperature (22° C.) for five minutes or at very low temperatures such as 2° C. in 2 microliters. It is also preferred that hybridization between nucleic acids be facilitated using buffers such as saline, Tris-EDTA (TE), Tris-HCl and other aqueous solutions, certain reagents and chemicals. Preferred examples of these reagents include single-stranded binding proteins such as Rec A protein, T4 gene 32 protein, E. coli single-stranded binding protein, and major or minor nucleic acid groove binding proteins. Preferred examples of other reagents and chemicals include divalent ions, polyvalent ions, and intercalating substances such as ethidium bromide, actinomycin D, psoralen, and angelicin.

[0051] The nucleotide sequence of the random portion of each probe is determinable by methods which are well-known in the art. Two methods for determining the sequence of the nucleic acid probe are by chemical cleavage, as disclosed by Maxam and Gilbert (1977), and by chain extension using ddNTPs, as disclosed by Sanger et al. (1977), both of which are herein specifically incorporated by reference. Alternatively, another method for determining the nucleotide sequence of a probe is to individually synthesize each member of a probe set. The entire set would comprise every possible sequence within the random portion or some smaller portion of the set. The method of the present invention could then be conducted with each member of the set. Another procedure would be to synthesize one or more sets of nucleic acid probes simultaneously on a solid support. Preferred examples of a solid support include a plastic, a ceramic, a metal, a resin, a gel, and a membrane. A more preferred embodiment comprises a two-dimensional or three-dimensional matrix, such as a gel, with multiple probe binding sites, such as a hybridization chip as described by Pevzner et al. (J. Biomol. Struc. & Dyn. 9:399-410, 1991), and by Maskos and Southern (Nuc. Acids Res. 20:1679-84, 1992), both of which are herein specifically incorporated by reference. Nucleic acids are bound to the solid support by covalent binding such as by conjugation with a coupling agent, or by non-covalent binding such as an electrostatic interaction or antibody-antigen coupling. Typical coupling agents include biotin/streptavidin, Staphylococcus aureus protein A/IgG antibody F_(c) fragment, and streptavidin/protein A chimeras (T. Sano and C. R. Cantor, Bio/Technology 9:1378-81, 1991).

[0052] Hybridization chips can be used to construct very large probe arrays which are subsequently hybridized with a target nucleic acid. Analysis of the hybridization pattern of the chip provides an immediate fingerprint identification of the target nucleotide sequence. Patterns can be manually or computer analyzed, but it is clear that positional sequencing by hybridization lends itself to computer analysis and automation. Algorithms and software have been developed for sequence reconstruction which are applicable to the methods described herein (R. Drmanac et al., J. Biomol. Struc. & Dyn. 5:1085-1102, 1991; P. A. Pevzner, J. Biomol. Struc. & Dyn. 7:63-73, 1989, both of which are herein specifically incorporated by reference).

[0053] Preferably, target nucleic acids are labeled with a detectable label. Label may be incorporated at a 5′ terminal site, a 3′ terminal site, or at an internal site within the length of the nucleic acid. Preferred detectable labels include a radioisotope, a stable isotope, an enzyme, a fluorescent chemical, a luminescent chemical, a chromatic chemical, a metal, an electric charge, or a spatial structure. There are many procedures whereby one of ordinary skill can incorporate detectable label into a nucleic acid. For example, enzymes used in molecular biology will incorporate radioisotope labeled substrate into nucleic acid. These include polymerases, kinases, and transferases. The labeling isotope is preferably, ³²P, ³⁵S, ¹⁴C, or ¹²⁵I.

[0054] Label may be directly or indirectly detected using scintillation fluid or a PhosphorImager, chromatic or fluorescent labeling, or mass spectrometry. Other, more advanced methods of detection include evanescent wave detection of surface plasmon resonance of thin metal film labels such as gold, by, for example, the BIAcore sensor sold by Pharmacia, or other suitable biosensors. Alternatively, the probe may be labeled and the target nucleic acid detected, identified and possibly sequenced from interaction with the labeled probe. For example, a labeled probe or array of probes may be fixed to a solid support. From an analysis of the binding observed after hybridization with a biological sample containing nucleic acid, the target nucleic acid is identified.

[0055] Another embodiment of the invention is directed to methods for determining a sequence of a nucleic acid comprising the steps of labeling the nucleic acid with a first detectable label at a terminal site, labeling the nucleic acid with a second detectable label at an internal site, identifying the nucleotide sequences of portions of the nucleic acid, determining the relationship of the nucleotide sequence portions to the nucleic acid by comparing the first detectable label and the second detectable label, and determining the nucleotide sequence of the nucleic acid. Fragments of target nucleic acids labeled both terminally and internally can be distinguished based on the relative amounts of each label within respective fragments. Fragments of a target nucleic acid terminally labeled with a first detectable label will have the same amount of label as fragments which include the labeled terminus. However, theses fragments will have variable amounts of the internal label directly proportional to their size and distance for the terminus. By comparing the relative amount of the first label to the relative amount of the second label in each fragment, one of ordinary skill is able to determine the position of the fragment or the position of the nucleotide sequence of that fragment within the whole nucleic acid.

[0056] Another embodiment of the invention is directed to methods for determining a nucleotide sequence by hybridization comprising the steps of creating a set of nucleic acid probes wherein each probe has a double-stranded portion, a single-stranded portion, and a random sequence within the single-stranded portion which is determinable, hybridizing a nucleic acid target which is at least party single-stranded to the set, ligating the hybridized target to the probe, and determining the nucleic sequence of the target which is hybridized to the single-stranded portion of any probe. This embodiment adds a step wherein the hybridized target is ligated to the probe. Ligation of the target nucleic acid to the complimentary probe increases fidelity of hybridization and allows for incorrectly hybridized target to be easily washed from correctly hybridized target (FIG. 11). More importantly, the addition of a ligation step allows for hybridiztions to be performed under a single set of hybridization conditions. For example, hybridization temperature is preferably between about 22-37^ OC., the salt concentration useful is preferably between about 0.05-0.5M, and the period of hybridization is between about 1-14 hours. This is not possible using the methodoligies of the current procedures which do not employ a ligation step and represents a very substantial improvement. Ligation can be accomplished using a eukaryotic derived or a prokaryotic derived ligase. Preferred is T4 DNA or RNA ligase. Methods for use of these and other nucleic acid modifying enzymes are described in Current Protocols in Molecular Biology (F. M. Ausubel et al., editors, John Wiley & Sons, 1989), which is herein specifically incorporated by reference.

[0057] There are a number of distinct advantages to the incorporation of a ligation step. First and foremost is that one can use identical hybridization conditions for hybridization. Variation of hybridization conditions due to base composition are no longer relevant as nucleic acids with high A/T or G/C content ligate with equal efficiency. Consequently, discrimination is very high between matches and mismatches, much higher than has been achieved using other methodologies such as Southern (1989) wherein the effects of G/C content were only somewhat neutralized in high concentrations of quarternary or tertiary amines (e.g., 3M tetramethyl ammonium chloride in Drmanac et al., 1993).

[0058] Another embodiment of the invention is directed to methods for determining a nucleotide sequence by hybridization which comprises the steps of creating a set of nucleic acid probes wherein each probe has a double-stranded portion, a single-stranded portion, and a random sequence within the single-stranded portion which is determinable, hybridizing a target nucleic acid which is at least partly single-stranded to the set of nucleic acid probes, enzymatically extending a strand of the probe using the hybridized target as a template, and determining the nucleotide sequence of the single-stranded portion of the target nucleic acid. This embodiment of the invention is similar to the previous embodiment, as broadly described herein, and includes all of the aspects and advantages described therein. An alternative embodiment also includes a step wherein hybridized target is ligated to the probe. Ligation increases the fidelity of the hybridization and allows for a more stringent wash step wherein incorrectly hybridized, unligated target can be removed and further, allows for a single set of hybridization conditions to be employed. Most nonligation techniques including Southern (1989), Drmanac et al. (1993), and Khrapko et al. (1989 and 1991), are only accurate, and only marginally so, when are hybriizations are performed under optimal conditions which vary with the G/C content of each interaction. Preferable condiions comprise a hybridization temperature of between about 22-37^ OC., a salt concentration of betwen about 0.05-0.5 M, and a hybridization period of between about 1-14 hours.

[0059] Hybridization produces either a 5′ overhang or a 3′ overhang of target nucleic acid. Where there is a 5′ overhang, a 3-hydroxyl is available on one strand of the probe from which nucleotide addition can be initiated. Preferred enzymes for this process include eukaryotic or prokaryotic polymerases such as T3 or T7 polymerase, Klenow fragment, or Taq polymerase. Each of these enzymes are readily available to those of ordinary skill in the art as are procedures for their use (Current Protocols in Molecular Biology).

[0060] Hybridized probes may also be enzymatically extended a predetermined length. For example, reaction condition can be established wherein a single dNTP or ddNTP is utilized as substrate. Only hybridized probes wherein the first nucleotide to be incorporated is complimentary to the target sequence will be extended, thus, providing additional hybridization fidelity and additional information regarding the nucleotide sequence of the target. Sanger (1977) or Maxam and Gilbert (1977) sequencing can be performed which would provide further target sequence data. Alternatively, hybridization of target to probe can produces 3′ extensions of target nucleic acids. Hybridized probes can be extended using nucleoside biphosphate substrates or short sequences which are ligated to the 5′ terminus.

[0061] Another embodiment of the invention is directed to a method for determining a nucleotide sequence of a target by hybridization comprising the steps of creating a set of nucleic acid probes wherein each probe has a double-stranded portion, a single-stranded portion, and a random nucleotide sequence within the single-stranded portion which is determinable, cleaving a plurality of nucleic acid targets to form fragments of various lengths which are at least partly single-stranded, hybridizing the single-stranded region of the fragments with the single-stranded region of the probes, identifying the nucleotide sequences of the hybridized portions of the fragments, and comparing the identified nucleotide sequences to determine the nucleotide sequence of the target. An alternative embodiment includes a further step wherein the hybridized fragments are ligated to the probes prior to identifying the nucleotide sequences of the hybridized portions of the fragments. As described heerin, the addition of a ligation step allows for hybridizations to be performed under a single set of hybridization conditions.

[0062] In these embodiments, target nucleic acid is partially cleaved forming a plurality of nucleic acid fragments of various lengths, a nested set, which is then hybridized to the probe. It is preferred that cleavage occurs by enzymatic, chemical or physical means. Preferred enzymes for partial cleavage are exonuclease III, S1 nuclease, DNase I, Bal 31, mung bean nuclease, P1 nuclease, lambda exonuclease, restriction endonuclease, and RNase I. Preferred means for chemical cleavage are ultraviolet light induced cleavage, ethidium bromide induced cleavage, and cleavage induced with acid or base. Preferred means for mechanical cleavage are shearing through direct agitation such as vortexing or multiple cycles of freeze-thawing. Procedures for enzymatic, chemical or physical cleavage are disclosed in, for example, Molecular Cloning. A Laboratory Manual (T. Maniatis et al., editors, Cold Spring Harbor 1989), which is herein specifically incorporated by reference.

[0063] Fragmented target nucleic acids will have a distribution of terminal sequences which is sufficiently broad so that the nucleotide sequence of the hybridized fragments will include the entire sequence of the target nucleic acid. A preferred method is wherein the set of nucleic acid probes is fixed to a solid support. A preferred solid support is a plastic, a ceramic, a metal, or magnetic substance, a resin, a film or other polymer, a gel, or a membrane, and it is more preferred that the solid support be a two-dimensional or three-dimensional matrix with multiple probe binding sites such as a hybridization chip as described by K. R. Khrapko et al. (J. DNA Sequencing and Mapping 1:357-88, 1991). It is also preferred wherein the target nucleic acid has a detectable label such as a radioisotope, a stable isotope, an enzyme, a fluorescent chemical, a luminescent chemical, a chromatic chemical, a metal, an electric charge, or a spatial structure.

[0064] As an extension of this procedure, it is also possible to use the methods herein described to determine the nucleotide sequence of one or more probes which hybridize with an unknown target sequence. For example, fragmented targets could be terminally or internally labeled, hybridized with a set of nucleic acid probes, and the hybridized sequences of the probes determined. This aspect may be useful when it is cumbersome to determine the sequence of the entire target and only a smaller region of that sequence is of interest.

[0065] Another embodiment of the invention is directed a method wherein the target nucleic acid has a first detectable label at a terminal site and a second detectable label at an internal site. The labels may be the same type of label or of different types as long as each can be discriminated, preferably by the same detection method. It is preferred that the first and second detectable labels are chromatic or fluorescent chemicals or molecules which are detectable by mass spectrometry. Using a double-labeling method coupled with analysis by mass spectrometry provides a very rapid and accurate sequencing methodology that can be incorporated in sequencing by hybridization and lends itself very well to automation and computer control.

[0066] Another embodiment of the invention is directed to methods for creating a nucleic acid probe comprising the steps of synthesizing a plurality of single-stranded first nucleic acids and an array of longer single-stranded second nucleic acids complimentary to the first nucleic acid with a random terminal nucleotide sequence, hybridizing the first nucleic acids to the second nucleic acids to form hybrids having a double-stranded portion and a single-stranded portion with the random nucleotide sequence within the single-stranded portion, hybridizing a single-stranded nucleic acid target to the hybrids, ligating the hybridized target to the first nucleic acid of the hybrid, isolating the second nucleic acid, and hybridizing the first nucleic acid of step with the isolated second nucleic acid to form a nucleic acid probe. Probes created in this manner are referred to herein as customized probes.

[0067] Preferred customized probe comprises a first nucleic acid which is about 15-25 nucleotides in length and the second nucleic acid is about 20-30 nucleotides in length. It is also preferred that the double-stranded portion contain an enzyme recognition site which allows for increased flexibility of use and facilitates cloning, should it at some point become desirable to clone one or more of the probes. It is also preferred if the customized probe is fixed to a solid support, such as, a plastic, a ceramic, a metal, a resin, a film or other polymer, a gel, or a membrane, or possibly a two- or three-dimensional array such as a chip or microchip.

[0068] Customized probes, created by the method of this invention, have a wide range of uses. These probes are, first of all, structurally useful for identifying and binding to only those sequences which are homologous to the overhangs. Secondly, the overhangs of these probes possess the nucleotide sequence of interest. No further manipulation is required to carry the sequence of interest to another structure. Therefore, the customized probes greatly lend themselves to use in, for example, diagnostic aids for the genetic screening of a biological sample.

[0069] Another embodiment of the invention is directed to arrays of nucleic acid probes wherein each probe comprises a double-stranded portion of length D, a terminal single-stranded portion of length S, and a random nucleotide sequence within the single-stranded portion of length R. Preferably, D is between about 3-20 nucleotides and S is between about 3-20 nucleotides and the entire array is fixed to a solid support which may be composed of plastics, ceramics, metals, resins, polymers and other films, gels, membranes and two-dimensional and three-dimensional matrices such as hybridization chips or microchips. Probe arrays are useful in sequencing and diagnostic applications when the sequence and/or position on a solid support of every probe of the array is known or is unknown. In either case, information about the target nucleic acid may be obtained and the target nucleic acid detected, identified and sequenced as described in the methods described herein. Arrays comprise 4^(R) different probes representing every member of the random sequence of length R, but arrays of less than 4^(R) are also encompassed by the invention.

[0070] Another embodiment of the invention is directed to method for creating probe arrays comprising the steps of synthesizing a first set of nucleic acids each comprising a constant sequence of length C at a 3′-terminus and a random sequence of length R at a 5′-terminus, synthesizing a second set of nucleic acids each comprising a sequence complimentary to the constant sequence of each of the first nucleic acid, and hybridizing the first set with the second set to create the array. Preferably, the nucleic acids of the first set are each between about 15-30 nucleotides in length and the nucleic acids of the second set are each between about 10-25 nucleotides in length. Also preferable is that C is between about 7-20 nucleotides and R is between about 3-10 nucleotides.

[0071] Arrays may comprise about 4^(R) different probes, but in certain applications, an entire array of every possible sequence is not necessary and incomplete arrays are acceptable for use. For example, incomplete arrays may be utilized for screening procedures of very rare target nucleic acids where nonspecific hybridization is not expected to be problematic. Further, every member of an array may not be needed when detecting or sequencing smaller nucleic acids where the chance of requiring certain combinations of nucleotides is so low as to be practically nonexistent. Arrays which are fixed to solid supports are expected to be most useful, although array in solution also have many applications. Solid supports which are useful include plastics such as microtiter plates, beads and microbeads, ceramics, metals where resilience is desired or magnetic beads for ease of isolation, resins, gels, polymers and other films, membranes or chips such as the two- and three-dimensional sequencing chips utilized in sequencing technology.

[0072] Alternatively, probe arrays may also be made which are single-stranded. These arrays are created, preferably on a solid support, basically as described, by synthesizing an array of nucleic acids each comprising a constant sequence of length C at a 3′-terminus and a random sequence of length R at a 5′-terminus, and fixing the array to a first solid support. Arrays created in this manner can be quickly and easily transformed into double-stranded arrays by the synthesis and hybridization of a set of nucleic acids with a sequence complimentary to the constant sequence of the replicated array to create a double-stranded replicated array. However, in their present form, single-stranded arrays are very valuable as templates for replication of the array.

[0073] Due to the very large numbers of probes which comprise most useful arrays, there is a great deal of time spent in simply creating the array. It requires many hours of nucleic acid synthesis to create each member of the array and many hours of manipulations to place the array in an organized fashion onto any solid support such as those described previously. Once the master array is created, replicated arrays or slaves, can be quickly and easily created by the methods of the invention which take advantage of the speed and accuracy of nucleic acid polymerases. Basically, methods for replicating an array of single-stranded probes on a solid support comprise the steps of synthesizing an array of nucleic acids each comprising a constant sequence of length C at a 3′-terminus and a random sequence of length R at a 5′-terminus, fixing the array to a first solid support, synthesizing a set of nucleic acids each comprising a sequence complimentary to the constant sequence, hybridizing the nucleic acids of the set with the array, enzymatically extending the nucleic acids of the set using the random sequences of the array as templates, denaturing the set of extended nucleic acids, and fixing the denatured nucleic acids of the set to a second solid support to create the replicated array of single-stranded probes.

[0074] Denaturation of the array can be performed by subjecting the array to heat, for example 90°-100° C. for 2-15 minutes, or highly alkaline conditions, such as by the addition of sodium hydroxide. Denaturation can also be accomplished by adding organic solvents, nucleic acid binding proteins or enzymes which promote denaturation to the array. Preferably, the solid supports are coated with a substance such as streptavidin and the nucleic acid reagents conjugated with biotin. Denaturation of the partial duplex leads to binding of the nucleic acids to the solid support.

[0075] Another embodiment of the invention is directed to methods for creating arrays of probes comprising the steps of synthesizing an array of single-stranded nucleic acids each containing a constant sequence at the 3′-terminus, another constant sequence at the 5′-terminus, and a random internal sequence of length R flanked by the cleavage site(s) of a restriction enzyme (on one or both sides), synthesizing an array of primers each compliementary to a portion of the constant sequence of the 3′-terminus, hybridizing the two arrays together to form hybrids, extending the sequence of each primer by polymerization using a sequence of the nucleic acid as a template, and cleaving the extended hybrids with the restriction enzyme to form an array of probes with a double-stranded portion at one terminus, a single-stranded portion containing the random sequence at the opposite terminus. Preferably, the nucleic acids are each between about 10-50 nucleotides in length and R is between about 3-5 nucleotides in length. Any of the restriction enzymes which produce a 3′- or 5′-overhang after cleavage are suitable for use to make the array. Some of the restriction enzymes which are useful in this regard, and their recognition sequences are depicted in Table 1. TABLE 1 Restriction Recognition Sequence Enzyme 5′-Overhang 3′-Overhang AlwN I 5′-CAG NNN↓CTG 3′-GTC↑NNN GAC Bbv I 5′-GCAGC(N)₈↓ 3′-CGTCG(N)₁₂↑ Bgl I 5′-GCCN NNN↓NGGC 3′-CGGN↑NNN NCCG BstX I 5′-CCAN NNNN↓NTGG 3′-GGTN↑NNNN NACC Dra III 5′-CAC NNN↓GTG 3′-GTG↑NNN CAC Fok I 5′-GGATG(N)₉↓ 3′-CCTAC(N)₁₃↑ Hga I 5′-GACGC(N)₅↓ 3′-CTGCG(N)₁₀↑ PflM I 5′-CCAN NNN↓NTGG 3′-GGTN↑NNN NACC SfaN I 5′-GCATC(N)₅↓ 3′-CGTAG(N)₉↑ Sfi I 5′-GGCCN NNN↓NGGCC 3′-CCGGN↑NNN NCCGG

[0076] Also prefered is that the array be fixed to a solid support such as a plastic, ceramic, metal, resin, polymer, gel, film, membrane or chip. Fixation can be accomplished by conjugating the reagents for synthesis with a specific binding protein or other similar substance and coating the surface of the support with the binding counterpart (e.g. biotin/streptavidin, F_(c)/protein A, nucleic acid/nucleic acid binding protein).

[0077] Alternatively, another similar method for creating an array of probes comprising the steps of synthesizing an array of single-stranded nucleic acids each containing a constant sequence at the 3′-terminus, another constant sequence at the 5′-terminus, and a random internal sequence of length R flanked by the cleavage site(s) of a restriction enzyme (on one or both sides), synthesizing an array of primers with a sequence complimentary the constant sequence at the 3′-terminus, hybridizing the two arrays together to form hybrids, enzymatically extending the primers using the nucleic acids as templates to form full-length hybrids, cloning the full-length hybrids into vectors such as plasmids or phage, cloning the plasmids into competent bacteria or phage, reisolating the cloned plasmid DNA, amplifying the cloned sequences by multiple polymerase chain reactions, and cleaving the amplified sequences with the restriction enzyme to form the array of probes with a double-stranded portion at one terminus and a single-stranded portion containing the random sequence at the opposite terminus. Using this method the array of probes may have 5′- or 3′-overhangs depending on the cleavage specificity of the restriction enzyme (e.g. Table 1). The array of probes may be fixed to a solid support such as a plastic, ceramic, metal, resin, polymer, film, gel, membranes and chip. Preferably, during PCR amplification, the reagent primers are conjugated with biotin which facilitates eventual binding to a streptavidin coated surface.

[0078] Another embodiment of the invention is directed to methods for using customized probes, arrays, and replicated arrays, as described herein, in diagnostic aids to screen biological samples for specific nucleic acid sequences. Diagnostic aids and methods for using diagnostic aids would be very useful when sequence information at a particular locus of, for example, DNA is desired. Single nucleotide mutations or more complex nucleic acid fingerprints can be identified and analyzed quickly, efficiently, and easily. Such an approach would be immediately useful for the detection of individual and family genetic variation, of inherited mutations such as those which cause a disease, DNA dependent normal phenotypic variation, DNA dependent somatic variation, and the presence of heterologous nucleic acid sequences.

[0079] Especially useful are diagnostic aids comprising probe arrays. These arrays can make the detection identification, and sequencing of nucleic acids from biological samples exceptionally rapid and allows one to obtain multiple pieces of information from a single sample after performing a single test. Methods for detecting and/or identifying a target nucleic acid in a biological sample comprise the steps of creating an array of probes fixed to a solid support as described herein, labeling the nucleic acid of the biological sample with a detectable label, hybridizing the labeled nucleic acid to the array and detecting the sequence of the nucleic acid from a binding pattern of the label on the array. These methods for creating probe arrays and for rapidly and efficiently replicating those arrays, such as for diagnostic aids, makes the manufacture and commercial application of large numbers of arrays a possibility.

[0080] As described, these diagnostic aids are useful to humans, other animals, and even plants for the detection of infections due to viruses, bacteria, fungi or yeast, and for the detection of certain parasites. These detection methods and aids are also useful in the feed and food industries and in the environmental field for the detection, identification and sequencing of nucleic acids associated with samples obtained from environmental sources and from manufacturing products and by-products.

[0081] Diagnostic aids comprise specific nucleic acid probes fixed to a solid support to which is added the biological sample. Hybridization of target nucleic acids is determined by adding a detectable label, such as a labeled antibody, which will specifically recognize only hybridized targets or, alternatively, unhybridized target is washed off and labeled target specific antibodies are added. In either case, appearance of label on the solid support indicates the presence of nucleic acid target hybridized to the probe and consequently, within the biological sample.

[0082] Customized probes may also prove useful in prophylaxis or therapy by directing a drug, antigen, or other substance to a nucleic acid target with which it will hybridize. The substance to be targeted can be bound to the probe so as not to interfere with possible hybridization. For example, if the probe was targeted to a viral nucleic acid target, an effective antiviral could be bound to the probe which will then be able to specifically carry the antiviral to infected cells. This would be especially useful when the treatment is harmful to normal cells and precise targeting is required for efficacy.

[0083] Another embodiment of the invention is directed to methods for creating a nucleic acid probe comprising the steps of synthesizing a plurality of single-stranded first nucleic acids and an array of longer single-stranded second nucleic acids complimentary to the first nucleic acid with a random terminal nucleotide sequence, hybridizing the first nucleic acids to the second nucleic acids to form hybrids having a double-stranded portion and a single-stranded portion with the random nucleotide sequence within the single-stranded portion, hybridizing a single-stranded nucleic acid target to the hybrids, ligating the hybridized target to the first nucleic acid of the hybrid, hybridizing the ligated hybrid with an array of oligonucleotides with random nucleotide sequences, ligating the hybridized oligonucleotide to the second nucleic acid of the ligated hybrid, isolating the second nucleic acid, and hybridizing another first nucleic acid with the isolated second nucleic acid to form a nucleic acid probe. Preferred is that the first nucleic acid is about 15-25 nucleotides in length, that the second nucleic acid is about 20-30 nucleotides in length, that the constant portion contain an enzyme recognition site, and that the oligonucleotides are each about 4-20 nucleotides in length. Probes may be fixed to a solid support such as a plastic, ceramic, a metal, a resin, a gel, or a membrane. It is preferred that the solid support be a two-dimensional or three-dimensional matrix with multiple probe binding sites such as a hybridization chip. Nucleic acid probes created by the method of the present invention are useful in a diagnostic aid to screen a biological sample for genetic variations of nucleic acid sequences therein.

[0084] Another embodiment of the invention is directed to a method for creating a nucleic acid probe comprising the steps of (a) synthesizing a plurality of single-stranded first nucleic acids and a set of longer single-stranded second nucleic acids complimentary to the first nucleic acid with a random terminal nucleotide sequence, (b) hybridizing the first nucleic acids to the second nucleic acids to form hybrids having a double-stranded portion and a single-stranded portion with the random nucleotide sequence in the single-stranded portion, (c) hybridizing a single-stranded nucleic acid target to the hybrids, (d) ligating the hybridized target to the first nucleic acid of the hybrid, (e) enzymatically extending the second nucleic acid using the target as a template, (f) isolating the extended second nucleic acid, and (g) hybridizing the first nucleic acid of step (a) with the isolated second nucleic acid to form a nucleic acid probe. It is preferred that the first nucleic acid is about 15-25 nucleotides in length, that the second nucleic acid is about 20-30 nucleotides in length, and that the double-stranded portion contain an enzyme recognition site. It is also preferred that the probe be fixed to a solid support, such as a plastic, ceramic, a metal, a resin, a gel, or a membrane. A preferred solid support is a two-dimensional or three-dimensional matrix with multiple probe binding sites, such as a hybridization chip. A further embodiment of the present invention is a diagnostic aid comprising the created nucleic acid probe and a method for using the diagnostic aid to screen a biological sample as herein described.

[0085] As an extension of this procedure, it is also possible to use the methods herein described to determine the nucleotide sequence of one or more probes which hybridize with an unknown target sequence. For example, Sanger dideoxynucleotide sequencing techniques could be used when enzymatically extending the second nucleic acid using the target as a template and labeled substrate, extended products could be resolved by polyacrylamide gel electrophoresis, and the hybridized sequences of the probes easily read off the gel. This aspect may be useful when it is cumbersome to determine the sequence of the entire target and only a smaller region of that sequence is of interest.

[0086] The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.

EXAMPLES Example 1

[0087] Manipulation of DNA in the solid state. Complexes between streptavidin (or avidin) and biotin represent the standard way in which much solid state DNA sequencing or other DNA manipulation is done, and one of the standard ways in which non-radioactive detection of DNA is carried out. Over the past few years streptavidin-biotin technology has expanded in several ways. Several years ago, the gene for streptavidin was cloned and sequenced (C. E. Argarana et al., Nuc. Acids Res. 14:1871, 1986). More recently, using the Studier T7 system, over-expression of the Protein in E. coli was achieved (T. Sano and C. R. Cantor, Proc. Natl. Acad. Sci. USA 87:142, 1990). In the last year, mutant streptavidins modified for improved solubility properties and firmer attachment to solid supports was also expressed (T. Sano and C. R. Cantor, Bio/Technology 9:1378-81, 1993). The most relevant of these is core streptavidin, (fully active protein with extraneous N- and C-terminal peptides removed) with 5 cysteine residues attached to the C-terminus. An active protein fusion of streptavidin to two IgG binding domains of staphylococcal A protein was also produced (T. Sano and C. R. Cantor, Bio/Technology 9:1378-81, 1991). This allowed biotinylated DNAs to be attached to specific Immunoglobulin G molecules without the need for any covalent chemistry, and it has led to the development of immuno-PCR, an exceedingly sensitive method for detecting antigens (T. Sano et al., Sci. 258:120-29, 1992).

[0088] A protein fusion between streptavidin and metallothionein was recently onstructed (T. Sano et al., Proc. Natl. Acad. Sci. USA, 1992). Both partners in this protein fusion are fully active and these streptavidin-biotin interactions are being used to develop new methods for purification of DNA, including triplex-mediated capture of duplex DNA on magnetic microbeads (T. Ito et al., Proc. Natl. Acad. Sci. USA 89:495-98, 1992) and affinity capture electrophoresis of DNA in agarose (T. Ito et al., G.A.T.A., 1992).

[0089] An examination of the potential advantages of stacking hybridization has been carried out by both calculations and pilot experiments. Some calculated T_(m)'s for perfect and mismatched duplexes are shown in FIG. 1. These are based on average base compositions. The calculations were preformed using the equations given by J. G. Wetmur (Crit. Rev. in Biochem. and Mol. Biol. 26:227-59, 1991). In the case of oligonucleotide stacking, these researchers assumed that the first duplex is fully formed under the conditions where the second oligomer is being tested; in practice this may not always be the case. It will, however, be the case for the configuration shown in FIG. 1. The calculations reveal a number of interesting features about stacking hybridization. Note that the binding of a second oligomer next to a pre-formed duplex provides an extra stability equal to about two base pairs. More interesting, still, is the fact that mispairing seems to have a larger consequence on stacking hybridization than it does on ordinary hybridization. This is consistent with the very large effects seen by K. R. Khrapko et al. (J. DNA Sequencing and Mapping 1:375-88, 1991) for certain types of mispairing. Other types of mispairing are less destabilizing, but these can be eliminated by requiring a ligation step. In standard SBH, a terminal mismatch is the least destabilizing event, and thus, leads to the greatest source of ambiguity or background. For an octanucleotide complex, an average terminal mismatch leads to a 6° C. lowering in T_(m). For stacking hybridization, a terminal mismatch on the side away from the pre-existing duplex, is the least destabilizing event. For a pentamer, this leads to a drop in T_(m) of 10° C. These considerations indicate that the discrimination power of stacking hybridization in favor of perfect duplexes might be greater than ordinary SBH.

Example 2

[0090] Terminal sequencing by positional hybridization. The basic sequencing by hybridization scheme is depicted in FIG. 2. It is different from any other because it uses a duplex oligonucleotide array with 3′-ended single-stranded overhangs. The duplex portion of each DNA shown is constant. Only the overhangs vary, and in principle an array of 4^(n) probes is needed to represent all possible overhangs of length n. The advantage of such an array is that it provides enhanced sequence stringency in detecting the 5′ terminal nucleotide of the target DNA because of base stacking between the preformed DNA duplex and the newly formed duplex.

[0091] One variable is the length of the single-stranded overhang. The shorter the overhang, the smaller the array of probes potentially useable. Overhangs of five and six have been successfully employed. The nature of the support surface to which the oligonucleotide is attached, the means of its attachment, and the length of the oligonucleotide duplex are also important variables. Initially one 5′ end-biotinylated strand of the probe duplex is attached to a solid surface. The technology is already well developed for the attachment of nucleic acids to solid supports, such as streptavidin-coated magnetic microbeads and membranes such as the thin gel system.

[0092] Another variable is the nucleic acid capacity of the immobilized spot of probe. This determines the detection sensitivity required and is also important where unlabeled DNA may be present that could hybridize competitively with the desired labeled DNA product. As depicted in FIG. 2A, the 3′ overhang of the array can detect the 3′-terminal sequence of the target DNA. These will derive from 5′-end labeled restriction fragments of known DNA sequence cut from vectors so that the target for the immobilized probe will either be at the 3′ end, just internal to it, or totally internal. In some subsequent examples, it does not matter whether hybridization is absolutely specific for the 3′ end.

[0093] Alternatively, positional sequencing by hybridization of the 5′-end single-stranded overhangs would be equally effective (FIG. 2B). This permits reading of the 5′ terminal sequence of the target DNA. However, this approach is not as versatile because it does not allow for the use of polymerases to enhance the length and accuracy of the sequence read.

Example 3

[0094] Preparation of model arrays. Following the scheme shown in FIG. 2, in a single synthesis, all 1024 possible single-stranded probes with a constant 18 base stalk followed by a variable 5 base extension can be created. The 18 base extension is designed to contain two restriction enzyme cutting sites. Hga I generates a 5 base, 5′ overhang consisting of the variable bases N₅. Not I generates a 4 base, 5′ overhang at the constant end of the oligonucleotide. The synthetic 23-mer mixture will be hybridized with a complimentary 18-mer to form a duplex which can then be enzymatically extended to form all 1024, 23-mer duplexes. These can be cloned by, for example, blunt end ligation, into a plasmid which lacks Not I sites. Colonies containing the cloned 23-base insert can be selected. Each should be a clone of one unique sequence. DNA minipreps can be cut at the constant end of the stalk, filled in with biotinylated pyrimidines, then cut at the variable end of the stalk, to generate the 5 base 5′ overhang. The resulting nucleic acid can be fractionated by Qiagen columns (nucleic acid purification columns) to discard the high molecular weight material, and the nucleic acid probe will then be attached to a streptavidin-coated surface. This procedure could easily be automated in a Beckman Biomec or equivalent chemical robot to produce many identical arrays of probes.

[0095] The initial array contains about a thousand probes. The particular sequence at any location in the array will not be known. However, the array can be used for statistical evaluation of the signal to noise ratio and the sequence discrimination for different target molecules under different hybridization conditions. Hybridization with known nucleic acid sequences allows for the identification of particular elements of the array. A sufficient set of hybridizations would train the array for any subsequent sequencing task. Arrays are partially characterized until they have the desired properties. For example, the length of the oligonucleotide duplex, the mode of its attachment to a surface, and the hybridization conditions used, can all be varied, using the initial set of cloned DNA probes. Once the sort of array that works best is determined, a complete and fully characterized array can then be constructed by ordinary chemical synthesis.

Example 4

[0096] Preparation of specific probe arrays. The major challenge for positional SBH, is to build real arrays of probes, and test the fraction of sequences that actually perform according to expectations. Base composition and base sequence dependence on the effectiveness of hybridization is probably the greatest obstacle to successful implementation of these methods. The use of enzymatic steps, where feasible, may simplify these problems, since, after all, the enzymes do manage to work with a wide variety of DNA sequences in vivo. With positional SBH, one potential trick to compensate for some variations in stability would be to allow the adjacent duplex to vary. Thus, for an A+T rich overhang, one could use a G+C rich stacking duplex, and vice versa.

[0097] Four methods for making arrays are tested and evaluated with two major objectives. The first is to produce, rapidly and inexpensively, arrays that will test some of the principles of positional SBH. The second is to develop effective methods for the automated preparation of full arrays needed for production sequencing via positional SBH. Since the first studies indicated that a five base overhang will be sufficient, arrays may only have to have 1024 members. The cost of making all of these compounds is actually quite modest. The constant portion of the probes can be made once, and then extended in parallel, by automated DNA synthesis methods. In the simplest case, this will require the addition of only 5 bases to each of 1024 compounds, which at typical chemical costs of $2 per base will amount to a total of about $10,000.

[0098] Moderately dense arrays can be made using a typical x-y robot to spot the biotinylated compounds individually onto a streptavidin-coated surface. Using such robots, it is possible to make arrays of 2×10⁴ samples in 100 to 400 cm² of nominal surface. T array should preferably fit in 10 cm², but even if forced, for unforeseen technical reasons, to compromise on an array ten times or even 50 times less dense, it will be quite suitable for testing the principles of and many of the variations on positional SBH. Commercially available streptavidin-coated beads can be adhered, permanently to plastics like polystyrene, by exposing the plastic first to a brief treatment with an organic solvent like triethylamine. The resulting plastic surfaces have enormously high biotin binding capacity because of the very high surface area that results. This will suffice for radioactively labeled samples.

[0099] For fluorescently labeled samples, the background scattering from such a bead-impregnated sample may interfere. In this case, a streptavidin-conjugated glass or plastic surface may be utilized (commercially available from Bios Products). Surfaces are made using commercially available amine-containing surfaces and using commercially available biotin-containing N-hydroxysuccinimide esters to make stable peptide conjugates. The resulting surfaces will bind streptavidin, at one biotin binding site (or at most two, but not more because the approximate 222 symmetry of the protein would preclude this), which would leave other sites available for binding to biotinylated oligonucleotides.

[0100] In certain experiments, the need for attaching oligonucleotides to surfaces may be circumvented altogether, and oligonucleotides attached to streptavidin-coated magnetic microbeads used as already done in pilot experiments. The beads can be manipulated in microtitre plates. A magnetic separator suitable for such plates can be used including the newly available compressed plates. For example, the 18 by 24 well plates (Genetix, Ltd.; USA Scientific Plastics) would allow containment of the entire array in 3 plates; this formate is well handled by existing chemical robots. It is preferable to use the more compressed 36 by 48 well formate, so that the entire array would fit on a single plate. The advantages of this approach for all the experiments are that any potential complexities from surface effects can be avoided, and already-existing liquid handling, thermal control, and imaging methods can be used for all the experiments. Thus, this allows the characterization of many of the features of positional SBH before having to invest the time and effort in fabricating instruments, tools and chips.

[0101] Lastly, a rapid and highly efficient method to print arrays has been developed. Master arrays are made which direct the preparation of replicas, or appropriate complementary arrays. A master array is made manually (or by a very accurate robot) by sampling a set of custom DNA sequences in the desired pattern and then transferring these sequences to the replica. The master array is just a set of all 1024-4096 compounds. It is printed by multiple headed pipettes and compressed by offsetting. A potentially more elegant approach is shown in FIG. 14. A master array is made and used to transfer components of the replicas in a sequence-specific way. The sequences to be transferred are designed so that they contain the desired 5 or 6 base 5′ variable overhang adjacent to a unique 15 base DNA sequence.

[0102] The master array consists of a set of streptavidin bead-impregnated plastic coated metal pins, each of which, at its tip, contains immobilized biotinylated DNA strands that consist of the variable 5 or 6 base segment plus the constant 15 base segment. Any unoccupied sites on this surface are filled with excess free biotin. To produce a replica chip, the master array is incubated with the complement of the 15 base constant sequence, 5′-labeled with biotin. Next, DNA polymerase is used to synthesize the complement of the 5 or 6 base variable sequence. Then the wet pin array is touched to the streptavidin-coated surface of the replica, held at a temperature above the T_(m) of the complexes on the master array. If there is insufficient liquid carryover from the pin array for efficient sample transfer, the replica array could first be coated with spaced droplets of solvent (either held in concave cavities, or delivered by a multiheaded pipettor). After the transfer, the replica chip is incubated with the complement of 15 base constant sequence to reform the double-stranded portions of the array. The basic advantage of this scheme, if it can be realized, is that the master array and transfer compounds are made only once, and then the manufacture of replica arrays should be able to proceed almost endlessly.

Example 5

[0103] DNA ligation to oligonucleotide arrays. Following the schemes shown in FIGS. 3A and 3B, E. coli and T4 DNA ligases can be used to covalently attach hybridized target nucleic acid to the correct immobilized oligonucleotide probe. This is a highly accurate and efficient process. Because ligase absolutely requires a correctly base paired 3′ terminus, ligase will read only the 3′-terminal sequence of the target nucleic acid. After ligation, the resulting duplex will be 23 base pairs long and it will be possible to remove unhybridized, unligated target nucleic acid using fairly stringent washing conditions. Appropriately chosen positive and negative controls demonstrate the power of this scheme, such as arrays which are lacking a 5′-terminal phosphate adjacent to the 3′ overhang since these probes will not ligate to the target nucleic acid.

[0104] There are a number of advantages to a ligation step. Physical specificity is supplanted by enzymatic specificity. Focusing on the 3′ end of the target nucleic also minimize problems arising from stable secondary structures in the target DNA. As shown in FIG. 3B, ligation can be used to enhance the fidelity of detecting the 5′-terminal sequence of a target DNA.

[0105] DNA ligases are also used to covalently attach hybridized target DNA to the correct immobilized oligonucleotide probe. Several tests of the feasibility of the ligation scheme shown in FIG. 3. Biotinylated probes were attached to streptavidin-coated magnetic microbeads, and annealed with a shorter, complementary, constant sequence to produce duplexes with 5 or 6 base single-stranded overhangs. One set of actual sequences used is shown in Example 14. ³²P-end labeled targets were allowed to hybridize to the Probes. Free targets were removed by capturing the beads with a magnetic separator. DNA ligase was added and ligation was allowed to proceed at various salt concentrations. The samples were washed at room temperature, again manipulating the immobilized compounds with a magnetic separator. This should remove non-ligated material. Finally, samples were incubated at a temperature above the T_(m) of the duplexes, and eluted single strand was retained after the remainder of the samples were removed by magnetic separation. The eluate at this point should consist of the ligated material. The fraction of ligation was estimated as the amount of ³²P recovered in the high temperature wash versus the amount recovered in both the high and low temperature washes. Results obtained are shown in FIG. 13. It is apparent that salt conditions can be found where the legation proceeds efficiently with perfectly matched 5 or 6 base overhangs, but not with G-T mismatches.

[0106] The results of a more extensive set of similar experiments are shown in Tables 2-4. Table 2 looks at the effect of the position of the mismatch and Table 3 examines the effect of base composition on the relative discrimination of perfect matches verses weakly destabilizing mismatches. These data demonstrate that: (1) effective discrimination between perfect matches and single mismatches occurs with all five base overhangs tested; (2) there is little if any effect of base composition on the amount of ligation seen or the effectiveness of match/mismatch discrimination. Thus, the serious problems of dealing with base composition effects on stability seen in ordinary SBH do not appear to be a problem for positional SBH; and (3) the worst mismatch positionis, as expected, the one distal from the phosphodiester bond formed in the ligation reaction. However, any mismatches that survive in this position will be eliminatd by a polymerase extension reaction, such as as described herein. provided that polymerase is used, like sequenase version 2, that has no 3′-endonuclease activity or terminal transferase activity; and (4) gel electrophoresis analysis has confirmed that the putative ligation products seen in these tests are indeed the actual products synthesized.

1 54 1 15 DNA Artificial Sequence Oligonucleotide 1 tcgagaacct tggct 15 2 18 DNA Artificial Sequence Oligonucleotide 2 ctactaggct gcgtagtc 18 3 23 DNA Artificial Sequence Oligonucleotide 3 gatgatccga cgcatcagag ctc 23 4 23 DNA Artificial Sequence Oligonucleotide 4 gatgatccga cgcatcagag ctt 23 5 23 DNA Artificial Sequence Oligonucleotide 5 gatgatccga cgcatcagag cta 23 6 23 DNA Artificial Sequence Oligonucleotide 6 gatgatccga cgcatcagag ccc 23 7 23 DNA Artificial Sequence Oligonucleotide 7 gatgatccga cgcatcagag ttc 23 8 23 DNA Artificial Sequence Oligonucleotide 8 gatgatccga cgcatcagaa ctc 23 9 23 DNA Artificial Sequence Oligonucleotide 9 gatgatccga cgcatcagag atc 23 10 23 DNA Artificial Sequence Oligonucleotide 10 gatgatccga cgcatcagag ctt 23 11 23 DNA Artificial Sequence Oligonucleotide 11 gatgatccga cgcatcagat atc 23 12 23 DNA Artificial Sequence Oligonucleotide 12 gatgatccga cgcatcagat att 23 13 24 DNA Artificial Sequence Oligonucleotide 13 gatgatccga cgcatcagag ctct 24 14 24 DNA Artificial Sequence Oligonucleotide 14 gatgatccga cgcatcagag ttct 24 15 41 DNA Artificial Sequence Oligonucleotide 15 gtcgacagtt gacgctacca ynnnnrtggt ctagagctag c 41 16 41 DNA Artificial Sequence Oligonucleotide 16 ctcgagagtt gacgctacca rnnnnytggt ctagacccgg g 41 17 12 DNA Artificial Sequence Primer 17 gctagctcta ga 12 18 41 DNA Artificial Sequence Oligonucleotide 18 gctagctcta gaccaynnnn rtggtagcgt caactgtcga c 41 19 12 DNA Artificial Sequence Primer 19 cccgggtcta ga 12 20 41 DNA Artificial Sequence Oligonucleotide 20 cccgggtcta gaccarnnnn ytggtagcgt caactctcga g 41 21 12 DNA Artificial Sequence Oligonucleotide 21 ggtnnnnnna cc 12 22 12 DNA Artificial Sequence Oligonucleotide - Compliment strand 22 ggtnnnnnna cc 12 23 15 DNA Artificial Sequence Oligonucleotide 23 gacgcnnnnn nnnnn 15 24 15 DNA Artificial Sequence Oligonucleotide - Compliment Strand 24 nnnnnnnnnn gcgtc 15 25 25 DNA Artificial Sequence Oligonucleotide 25 gtcgacagtt gacgctacca ynnnn 25 26 21 DNA Artificial Sequence Oligonucleotide 26 rtggtagcgt caactgtcga c 21 27 20 DNA Artificial Sequence Oligonucleotide 27 gctagctcta gaccaynnnn 20 28 16 DNA Artificial Sequence Oligonucleotide 28 rtggtctaga gctagc 16 29 25 DNA Artificial Sequence Oligonucleotide 29 ctcgagagtt gacgctacca rnnnn 25 30 21 DNA Artificial Sequence Oligonucleotide 30 ytggtagcgt caactctcga g 21 31 20 DNA Artificial Sequence Oligonucleotide 31 cccgggtcta gaccarnnnn 20 32 16 DNA Artificial Sequence Oligonucleotide 32 ytggtctaga cccggg 16 33 20 DNA Artificial Sequence Oligonucleotide 33 tggtagcgtc aactgtcgac 20 34 15 DNA Artificial Sequence Oligonucleotide 34 tggtctagag ctagc 15 35 20 DNA Artificial Sequence Oligonucleotide 35 tggtagcgtc aactctcgag 20 36 15 DNA Artificial Sequence Oligonucleotide 36 tggtctagac ccggg 15 37 13 DNA Artificial Sequence Bbv I Recognition Sequence 37 gcagcnnnnn nnn 13 38 17 DNA Artificial Sequence Bbv I Recognition Sequence - Complment 38 nnnnnnnnnn nngctgc 17 39 11 DNA Artificial Sequence Bgl I Recognition Sequence 39 gccnnnnngg c 11 40 11 DNA Artificial Sequence Bgl I Recognition Sequence - Complement 40 gccnnnnngg c 11 41 12 DNA Artificial Sequence BstX I Recognition Sequence 41 ccannnnnnt gg 12 42 12 DNA Artificial Sequence BstX I Recognition Sequence - Complement 42 ccannnnnnt gg 12 43 9 DNA Artificial Sequence Dra III Recognition Sequence 43 cacnnngtg 9 44 9 DNA Artificial Sequence Dra III Recognition Sequence - Complement 44 cacnnngtg 9 45 14 DNA Artificial Sequence Fok I Recognition Sequence 45 ggatgnnnnn nnnn 14 46 18 DNA Artificial Sequence Fok I Recognition Sequence - Complement 46 nnnnnnnnnn nnncatcc 18 47 10 DNA Artificial Sequence Hga I Recognition Sequence 47 gacgcnnnnn 10 48 15 DNA Artificial Sequence Hga I Recognition Sequence - Complement 48 nnnnnnnnnn gcgtc 15 49 11 DNA Artificial Sequence PflM I Recognition Sequence 49 ccannnnntg g 11 50 11 DNA Artificial Sequence PflM I Recognition Sequence - Complement 50 ccannnnntg g 11 51 10 DNA Artificial Sequence SfaN I Recognition Sequence 51 gcatcnnnnn 10 52 14 DNA Artificial Sequence SfaN I Recognition Sequence - Complement 52 nnnnnnnnng atgc 14 53 13 DNA Artificial Sequence Sif I Recognition Sequence 53 ggccnnnnng gcc 13 54 13 DNA Artificial Sequence Sif I Recognition Sequence - Complement 54 ggccnnnnng gcc 13 

We claim:
 1. A method for creating an array of probes comprising the steps of: a) synthesizing a first set of nucleic acids each comprising a constant sequence of length C at 3′-terminus and a random sequence of length R at a 5′-terminus; b) synthesizing a second set of nucleic acids each comprising a sequence complimentary to the constant sequence of each of the first nucleic acid; and c) hybridizing the first set with the second set to create the array.
 2. The method of claim 1 wherein the nucleic acids of the first set are each between about 15-30 nucleotides in length and the nucleic acids of the second set are each between about 10-25 nucleotides in length.
 3. The method of claim 1 wherein C is between about 7-20 nucleotides and R is between about 3-5 nucleotides.
 4. The method of claim 1 wherein the array comprises about 4^(R) different probes.
 5. The method of claim 1 wherein the array is fixed to a solid support and the solid support is selected from the group consisting of plastics, ceramics, metals, resins, gels, membranes and chips.
 6. An array of probes created by the method of claim
 1. 7. A method for creating an array of probes fixed to a solid support comprising the steps of: a) synthesizing a first set of nucleic acids each comprising a constant sequence of length C at a 3′-terminus and a random sequence of length R at a 5′-terminus; b) fixing the first set to the solid support; c) synthesizing a second set of nucleic acids each comprising a sequence complimentary to the constant region of the first set; and d) hybridizing the nucleic acids of the first set with the second set to create the array.
 8. A method for creating an array of probes comprising the steps of: a) synthesizing an array of single-stranded nucleic acids each containing a constant sequence at the 3′-terminus, another constant sequence at the 5′-terminus, and a random internal sequence of length R flanked by the cleavage sites of a restriction enzyme; b) synthesizing an array of primers each compliementary to a portion of the constant sequence of the 3′-terminus, hybridizing the two arrays together to form hybrids; c) extending the sequence of each primer by polymerization using a sequence of the nucleic acid as a template; and d) cleaving the extended hybrids with the restriction enzyme to form an array of probes with a double-stranded portion at one terminus, a single-stranded portion containing the random sequence at the opposite terminus.
 9. The method of claim 8 wherein the nucleic acids are each between about 10-50 nucleotides in length.
 10. The method of claim 8 wherein R is between about 3-5 nucleotides in length.
 11. The method of claim 8 wherein the restriction enzyme is selected from the group consisting of restriction enzymes which produce 5′-overhangs and restriction enzymes which produce 3′-overhangs.
 12. The method of claim 8 wherein the array of probes is fixed to a solid support and the solid support which is selected from the group consisting of plastics, ceramics, metals, resins, gels, membranes and chips.
 13. An array of probes created by the method of claim
 8. 14. A method for creating an array of probes comprising the steps of: a) synthesizing an array of single-stranded nucleic acids each containing a constant sequence at the 3′- terminus, another constant sequence at the 5′-terminus, and a random internal sequence of length R flanked by the cleavage sites of a restriction enzyme; b) synthesizing an array of primers with a sequence complimentary to the constant sequence at the 3′-terminus; c) hybridizing the two arrays together to form hybrids; d) enzymatically extending the primers using the nucleic acids as templates to form full-length hybrids; e) cloning the full-length hybrids into vectors; f) amplifying the cloned sequences by multiple polymerase chain reactions; and g) cleaving the amplified sequences with the restriction enzyme to form the array of probes with a double-stranded portion at one terminus and a single-stranded portion containing the random sequence at the opposite terminus.
 15. The method of claim 14 wherein the array of probes have 5′- or 3′-overhangs.
 16. The method of claim 14 wherein the array of probes is fixed to a solid support and the solid support is selected from the group consisting of plastics, ceramics, metals, resins, polymers, films, gels, membranes and chips.
 17. An array of probes created by the method of claim
 14. 18. A method for detecting a nucleic acid in a biological sample comprising the steps of: a) creating an array of probes fixed to a solid support according to the method of claim 7; b) labeling the nucleic acid of the biological sample with a detectable label; c) hybridizing the labeled nucleic acid to the array; and d) detecting the sequence of the nucleic acid from a binding pattern of the label on the array.
 19. A method for identifying a target nucleic acid in a biological sample comprising the steps of: a) creating an array of probes fixed to a solid support according to the method of claim 7; b) labeling the target of the biological sample with a detectable label; c) hybridizing the labeled target to the array; and d) identifying the target from a binding pattern of the label on the array.
 20. The method of claim 19 wherein the detectable label is selected from the group consisting of radioisotopes, stable isotopes, enzymes, fluorescent and luminescent chemicals, chromatic chemicals, metals, electric charges, and spatial chemicals.
 21. The method of claim 19 wherein the nucleic acid identified is selected from the group consisting of nucleic acids derived from viruses, bacteria, parasites, fungi and yeast.
 22. The method of claim 19 wherein the binding pattern is a nucleic acid fingerprint.
 23. A diagnostic aid for detecting a target nucleic acid in a biological sample comprising the array of claim 19 a solid support on which the array is fixed, a detectable label, and the biological sample.
 24. The method of claim 19 wherein the biological sample is selected from the group consisting of samples of animal tissue, environmental substances, and manufacturing products and by-products.
 25. The method of claim 24 wherein the animal tissue is obtained from a human.
 26. The method of claim 19 further comprising the step of purifying the target nucleic acids identified.
 27. A method for replicating an array of single-stranded probes on a solid support comprising the steps of: a) synthesizing an array of nucleic acids each comprising a constant sequence of length C at a 3′-terminus and a random sequence of length R at a 5′-terminus; b) fixing the array to a first solid support; c) synthesizing a set of nucleic acids each comprising a sequence complimentary to the constant sequence; d) hybridizing the nucleic acids of the set with the array; e) enzymatically extending the nucleic acids of the set using the random sequences of the array as templates; f) denaturing the set of extended nucleic acids; and g) fixing the denatured nucleic acids of the set to a second solid support to create the replicated array of single-stranded probes.
 28. The method of claim 27 wherein the nucleic acids of the set are conjugated with biotin and the second solid support comprises streptavidin.
 29. The method of claim 27 wherein the nucleic acids of the array are between about 15-30 nucleotides in length and the nucleic acids of the set are between about 10-25 nucleotides in length.
 30. The method of claim 27 wherein C is between about 7-20 nucleotides and R is between about 3-5 nucleotides.
 31. The method of claim 27 wherein the solid support is selected from the group consisting of plastics, ceramics, metals, resins, gels, membranes and chips.
 32. The method of claim 27 wherein the nucleic acids of the set are enzymatically extended with a DNA polymerase and one or more deoxynucleotide triphosphates.
 33. The method of claim 27 wherein denaturing is performed with heat, alkali, organic solvents, binding proteins, enzymes, salts or combinations thereof.
 34. A replicated array of single-stranded probes made by the method of claim
 27. 35. The method of claim 27 further comprising the step of hybridizing the replicated array with a second set of nucleic acids complimentary to the constant sequence of the replicated array to create a double-stranded replicated array.
 36. A replicated array of double-stranded probes made by the method of claim
 35. 37. A method for creating a probe comprising the steps of: a) synthesizing a plurality of first nucleic acids and a plurality of second nucleic acids comprising a random terminal sequence and a sequence complimentary to a sequence of the first nucleic acids; b) hybridizing the first nucleic acids with the second to form partial duplexes; c) hybridizing a target nucleic acid to the partial duplexes; d) ligating the hybridized target to the first nucleic acid of the partial duplexes; e) isolating the second nucleic acid from the ligated duplexes; and f) synthesizing a plurality of third nucleic acids each complimentary to the constant sequence of the second nucleic acid and hybridizing the third nucleic acids with the isolated second nucleic acids to create a probe.
 38. The method of claim 37 wherein the first nucleic acids are each between about 15-25 nucleotides in length and the second nucleic acids are each between about 20-30 nucleotides in length.
 39. The method of claim 37 wherein the target is hybridized to the partial duplexes under a single set of hybridization conditions.
 40. The method of claim 39 wherein the hybridization conditions comprise a temperature of between about 22-37^ OC., a salt concentration of between about 0.05-0.2 M, and a time period of between about 1-14 hours.
 41. The method of claim 37 wherein a double-stranded portion of the partial duplex contains an enzyme recognition site.
 42. A probe created by the method of claim
 37. 43. The probe of claim 42 which is fixed to a solid support and the solid support is selected from the group consisting of plastics, ceramics, metals, resins, gels, membranes and chips.
 44. A diagnostic aid for the detection of a target nucleic acid in a biological sample comprising the probe of claim 42, a solid support on which the probe is fixed, a detectable label, and the biological sample.
 45. A method for creating a probe comprising the steps of: a) synthesizing a plurality nucleic acids and a plurality of second nucleic acids each comprising a random terminal sequence and a sequence complimentary to the sequence of the first nucleic acids; b) hybridizing the first nucleic acids with the second nucleic acids to form partial duplexes having a double-stranded portion and a single-stranded portion with the random sequence within the single-stranded portion; c) hybridizing a target nucleic acid to the partial duplexes; d) ligating the hybridized target to the first nucleic acid of the partial duplex; e) hybridizing the ligated target with a set of oligonucleotides comprising random sequences; f) ligating the hybridized oligonucleotide to the second nucleic acid; g) isolating the oligonucleotide ligated second nucleic acid; and h) synthesizing another plurality of first nucleic acids and hybridizing the first nucleic acids with the isolated second nucleic acid to create the probe.
 46. The method of claim 45 wherein the first nucleic acids are each between about 15-25 nucleotides in length, the second nucleic acids are each between about 20-30 nucleotides in length, and the oligonucleotides are each between about 4-20 nucleotides in length.
 47. The method of claim 45 wherein the target is hybridized to the partial duplexes under a single set of hybridization conditions.
 48. The method of claim 45 wherein the hybridization conditions comprise a temperature of between about 22-37^ OC., a salt concentration of between about 0.05-0.2 M, and a time period of between about 1-14 hours.
 49. The method of claim 45 wherein the partial duplexes contain an enzyme recognition site.
 50. A nucleic acid probe created by the method of claim
 45. 51. The nucleic acid probe of claim 50 which is fixed to a solid support selected from the group consisting of plastics, ceramics, metals, resin, gels, membranes and chips.
 52. A diagnostic aid for the detection of a target nucleic acid in a biological sample comprising the probe of claim 45, a solid support on which the probe is fixed, a detectable label, and the biological sample.
 53. A method for creating a probe comprising the steps of: a) synthesizing a plurality of first nucleic acids and a plurality of second nucleic acids comprising a random terminal sequence and a sequence complimentary to a sequence of the first nucleic acid; b) hybridizing the first nucleic acids to the second nucleic acids to form partial duplexes having a double-stranded portion and a single-stranded portion with the random nucleotide sequence within the single-stranded portion; c) hybridizing a target nucleic acid to the partial duplexes; d) ligating the hybridized target to the first nucleic acid of the partial duplex; e) enzymatically extending the second nucleic acid using the target as a template; f) isolating the extended second nucleic acid; and g) synthesizing another first nucleic acid and hybridizing the first nucleic acid with the isolated and extended second nucleic acid to create a probe.
 54. The method of claim 53 wherein the first nucleic acids are each between about 15-25 nucleotides in length and the second nucleic acids are each between about 20-30 nucleotides in length.
 55. The method of claim 53 wherein the target is hybridized to the partial duplexes under a single set of hybridization conditions.
 56. The method of claim 55 wherein the hybridization conditions comprise a temperature of between about 22-37^ OC., a salt concentration of between about 0.05-0.2 M, and a time period of between about 1-14 hours.
 57. The method of claim 53 wherein the double-stranded portion contains an enzyme recognition site.
 58. The method of claim 53 wherein the target nucleic acid is obtained from a biological sample selected from the group consisting of samples of animal tissue, environmental substances, and manufacturing products and by-products.
 59. A nucleic acid probe created by the method of claim
 53. 60. The nucleic acid probe of claim 59 which is fixed to a solid support and the solid support is selected from the group consisting of plastics, ceramics, metals, resins, gels, membranes and chips.
 61. A diagnostic aid for the detection of a target nucleic acid in a biological sample comprising the nucleic acid probe of claim 59, a solid support on which the probe is fixed, a detectable label and the biological sample.
 62. An array of 4^(R) different nucleic acid probes wherein each probe comprises a double-stranded portion of length D, a terminal single-stranded portion of length S, and a random nucleotide sequence within the single-stranded portion of length R.
 63. The array of claim 62 wherein D is between about 3-20 nucleotides and S is between about 3-20 nucleotides.
 64. The array of claim 62 which is fixed to a solid support wherein the solid support is selected from the group consisting of plastics, ceramics, metals, resins, gels, membranes and two-dimensional and three-dimensional matrices. 